人工感染猪胎儿猪细小病毒的强毒复壮及其在猪肾原代细胞上的增殖

猪细小病毒(PPV)强毒，在1～5日龄仔猪肾原代细胞上传代，传至20代左右毒力下降。为保持PPV强毒的毒力，我们用一头妊娠母猪(妊娠48d)做剖腹术。用大剂量的PPV细胞培养物注入猪胎儿的羊膜腔内，人工感染猪胎儿，病毒通过猪胎儿体内的组织进行复制，使毒力增强。术后12d迫杀母猪，取胎儿分离PPV强毒。     

Pathogenicity of a skin isolate of porcine parvovirus in swine fetuses

The pathogenic properties of a skin isolate of porcine parvovirus (PPV), designated Kresse isolate, were compared with NADL-8 isolate, a prototype isolate of PPV, by in utero inoculation of mid-term and late-term gestation swine fetuses. Fetuses from pregnant sows of mid-gestation were inoculated with either NADL-8 or Kresse virus. Both isolates were highly pathogenic to mid-gestation fetuses. In contrast, dramatic differences in pathogenicity between these 2 isolates were observed in fetuses inoculated late in gestation. Such fetuses from each of 4 sows were inoculated with NADL-8 or Kresse virus isolate and sacrificed at 10, 18, 21, or 23 days postinoculation (PI). NADL-8-inoculated fetuses were grossly normal. The pathogenic effects of Kresse isolate were evident by gross pathology in fetuses collected at 18, 21, and 23 days PI, but not at 10 days PI. Hemagglutination (HA) and fluorescent antibody (FA) methods were used to identify virus in various tissues of late-gestation fetuses collected at 10 and 21 days PI. At 10 days PI, HA antigens were detected only in livers of NADL-8-inoculated fetuses, but in all tissues examined of Kresse-inoculated fetuses, including the brain. PPV specific fluorescence was demonstrated in tissues of fetuses inoculated with NADL-8 and Kresse virus. The major difference was that virus antigen was found in the brains of fetuses inoculated with Kresse virus, but not in NADL-8 infected fetuses. At 21 days PI, HA antigen was not detected in any of the tissues of fetuses inoculated with NADL-8 virus, with PPV specific fluorescence by FA being found only in the kidney. However, fetuses inoculated with Kresse virus displayed HA antigen in liver and PPV-specific fluorescence in all tissues tested including the brain. Both isolates induced similar antibody responses, 1:128 to 1:256 at 10 days and 1:512 to 1:1024 at 21 days PI. In addition, immunoglobulin G (IgG) deposits were demonstrated in kidneys and skin of fetuses inoculated with Kresse virus and IgM in brain, but not in tissues from fetuses inoculated with NADL-8 virus.     

Experimental in utero infection of pig foetuses with porcine parvovirus (PPV).

Pig foetuses of various gestational ages were exposed to experimental infection with porcine parvovirus (PPV) in utero. Inoculation of 40-, 50- and 60-day-old foetuses with PPV caused foetal death and mummification and spread of the infection to non-inoculated foetuses. Inoculation at 80 and 100 days gestation caused pathological lesions of various degrees whereas spread of infection occurred only sporadically. Serological examinations of foetuses of different ages suggest that immunocompetence for PPV develops before 70 days gestation. The present results strongly indicate that intrauterine spread of PPV is a route of transmission of this virus between pig foetuses.     

Pathogenesis of in utero infection: experimental infection of eight- and ten-week-old porcine fetuses with porcine parvovirus.

Selected numbers of fetuses in each of 4 pregnant gilts were exposed to a porcine parvovirus by injecting the virus into the allantoic fluid at gestation day 56 or 70. The fetuses were examined on postexposure day 7 or 14. When pregnancy was terminated, 2 of 15 exposed fetuses were dead. Several fetuses tested at post-exposure day 14 had hemagglutination-inhibiting antibodies to porcine parvovirus. Virus was detected most frequently and in highest concentration in the parenchymatous organs of thorax and abdomen of fetuses exposed on gestation day 56. A small amount of antigen was in neurons and capillary endothelium of cerebral and cerebellar cortexes.     

纳米铝胶佐剂增强猪细小病毒灭活疫苗猪体免疫的效果

将20头35日龄断奶仔猪随机分为4组,将制备的纳米铝胶佐剂猪细小病毒(Porcine parvovirus,PPV)灭活疫苗肌肉注射免疫接种,并以常规铝胶佐剂PPV灭活疫苗组、市售PPV油乳剂灭活疫苗组为疫苗对照,生理盐水组做阴性对照,应用HI和ELISA检测接种猪的体液免疫水平,流式细胞仪检测免疫猪的细胞免疫水平。体液免疫检测结果显示,PPV纳米铝胶佐剂疫苗组能显著提高机体产生抗体的速度,在首免后第2周产生有效保护抗体水平显著高于油乳剂疫苗和常规铝胶佐剂疫苗组(P0.05),在第3周其抗体水平达到峰值;细胞免疫检测结果显示,3种疫苗均能诱导机体产生细胞免疫应答,但各组之间差异不显著(P0.05)。结果表明,以纳米铝胶为佐剂制备的猪细小病毒灭活疫苗,能显著提高机体的免疫水平,并较早刺激机体产生抗体,具有较好的免疫保护效果。     

Detection of antibodies against porcine parvovirus in swine sera by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of antibody against porcine parvovirus in swine sera. The antigen used for the assay was partially-purified virus treated with fluorocarbon and shown to contain 7 proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Of these proteins 83-, 64- and 60-K proteins reacted in Western immunoblotting with swine serum after infection with porcine parvovirus. Antibody responses were demonstrated by ELISA in pigs subcutaneously-infected with porcine parvovirus as by hemagglutination-inhibition (HI) test and Western immunoblotting reaction with the 83-, 64- and 60-K viral proteins. The results of ELISA on random swine-serum samples were well-correlated with those of the HI test. These findings indicate the usefulness of the ELISA as a serological tool for porcine parvovirus infection.     

Persistence of porcine reproductive and respiratory syndrome virus infection in a swine operation.

A herd of Quebec seedstock pigs experienced in early 1992 a typical outbreak of porcine reproductive and respiratory syndrome (PRRS) associated with lesions of interstitial, proliferative and necrotizing pneumonia in weaned piglets. The nature of the infection was confirmed by serology using indirect immunofluorescence (IIF) and virus isolation in primary cultures of porcine alveolar macrophages (PAM). Farm production recovered after eight weeks of losses. In order to evaluate the persistence of infection in the herd, five SPF-piglets were introduced in two different sections of the PRRS-affected barn four months after the disappearance of clinical symptoms, and two others were placed in a neighboring building with apparently healthy farrow-to-finnish pigs. Clinical signs, body temperature, humoral immune response, virological and histopathological findings were recorded over a 42-day period. Clinical signs were evident in all of the sentinels and prolonged fever (> or = 40 degrees C) was recorded one day post-exposure (PE). Antibody titers to PRRS virus could be detected by IIF on PAM seven days PE, and reached 1:1024 by day 21 PE. Three of the sentinels developed significant virus neutralizing antibody titers (> 1:8 to < or = 1:128) by day 35 PE. In all cases, the virus could be isolated from the serum between day 7 and 42 PE. Thus, the virus and specific antibodies coexisted for several weeks. Lesions of interstitial pneumonia was demonstrated in few animals. In experimental inoculation studies, the viral strain isolated from the sentinel pigs produced severe reproductive disorders in two sows inoculated at 95 days of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excretion of porcine parvovirus through the genital tract of boars

The putative binding of porcine parvovirus (PPV) to semen components in vitro was examined along with the shedding pattern of PPV in oronasally infected boars. Porcine parvovirus DNA was determined to be bound to spermatozoa that had been incubated in vitro with PPV and washed to remove loosely adherent virus. To determine whether PPV was shed in the semen, four 8-month-old boars, seronegative for PPV, were inoculated oronasally with a virulent strain of PPV. Prior to virus inoculation, a catheter was surgically implanted in the vas deferens for the purpose of collecting cauda epididymal semen free of extrinsic contamination. Epididymal semen specimens were collected prior to inoculation and daily thereafter for 21 days. A fifth boar was inoculated oronasally with PPV, but semen was collected by electroejaculation twice weekly for an equal period of time. Reproductive glands and semen specimens from all boars were examined by nucleic acid hybridization for the presence of viral DNA. All boars seroconverted to PPV, as evidenced by serum antibody titers ranging from 512 to 8,192 hemagglutinating inhibition units/50 microliters. Porcine parvovirus DNA was detected in epididymal semen of 3 of 4 catheterized boars on postinoculation days 5 through 9, but not in semen obtained by electroejaculation. Viral DNA was consistently detected in tissue samples collected on postinoculation days 8 and 21 from the scrotal lymph nodes (4 of 5 boars) and epididymides (3 of 5 boars).     

Prenatal and preweaning deaths caused by pseudorabies virus and porcine parvovirus in a swine herd

Sequential outbreaks of pseudorabies virus and porcine parvovirus infections were documented at a swine farm in southern Minnesota. Data for the prevalence of mummified fetuses born and the preweaning mortality were recorded over a 3-year-period. The farm was a farrow-to-finish facility, with breeding females housed in 4 groups according to their stage of pregnancy. The herd consisted of approximately 130 breeding females in December 1981, and expanded to 220 females during the 12 months of 1982. Excluding the outbreaks, the mean preweaning mortality was 20.43% (SE 1.59) and the number of mummified fetuses per litter was 0.19 (SE 0.01). An outbreak of porcine parvovirus infection caused the preweaning mortality and number of mummified fetuses to increase to 50% and 4.10 per litter, respectively. Two outbreaks of pseudorabies 27 months apart, caused the preweaning mortality to increase to 95% and 82%, and the number of mummified fetuses to increase to 0.96 and 1.25 mummified fetuses per litter, respectively. The increase in mummification was observed 1 month after the increase in preweaning mortality caused by pseudorabies virus infections, whereas the increase in mummification and preweaning mortality was simultaneous with porcine parvovirus infections.     

Experimental in utero inoculation of late-term swine fetuses with porcine circovirus type 2.

All 37 fetuses of 3 laparotomized pregnant sows at 86, 92, and 93 days of gestation were inoculated intramuscularly through the uterine wall with porcine circovirus type 2 (PCV-2). The sows were allowed to farrow, and blood and tissue samples were collected from their piglets before and after suckling colostrum. Thirteen fetuses from 2 sows at 90 and 103 days of gestation were used as controls. Of the 37 PCV-2 inoculated fetuses, 24 were grossly normal and 13 were mummified, stillborn, or weak-born at farrowing. Infection with PCV-2 was demonstrated in various tissues of grossly normal and abnormal fetuses by virus isolation, polymerase chain reaction, and immunohistochemical methods. Antibodies specific to PCV-2 were also detected from the sera or thoracic fluids of abnormal fetuses and unsuckled normal pigs. No evidence of PCV-2 infection was found in any control fetuses. The present results confirm previous findings that PCV-2 can infect late-term swine fetuses and may cause reproductive abnormalities.     

Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus

One-day-old gnotobiotic piglets were inoculated intranasally with in vitro passaged porcine circovirus 1 (PCV-1), PCV-2, and porcine parvovirus (PPV) alone or in combination (PCV-1/PCV-2, PCV-1/PPV, and PCV-2/PPV). Piglets were evaluated for 1) the development of porcine postweaning multisystemic wasting syndrome (PMWS), 2) distribution of viral antigens by immunochemistry, and 3) viremia and the presence of viral DNA in nasal and ocular secretions and feces. All single agent-infected piglets and piglets infected with PCV-1/PCV-2 or PCV-1/PPV were clinically asymptomatic. They were transiently viremic and seroconverted to homologous virus(es). At termination of the study on postinfection day (PID) 35, microscopic lesions were restricted to focal inflammatory cell infiltrates in livers and myocardia. One piglet given PCV-1/PPV was PPV viremic for 2 weeks after infection and had lymphangiectasia of the spiral and descending colon associated with granulomatous inflammation. All four PCV-2/PPV-inoculated piglets developed PMWS, characterized by sudden onset of depression and anorexia, icterus, and submucosal edema. One piglet became moribund on PID 27, and the remaining three piglets were euthanatized between PID 27 and PID 30 because of severe disease. Lymph nodes were small and the livers were mottled. Disseminated angiocentric granulomatous inflammation was present in all tissues examined except the brain. Multiple lightly basophilic intracytoplasmic inclusion bodies were identified in macrophages and histiocytes. PCV-2 antigen was widely distributed within macrophages; PPV antigen was sparse. Hepatocellular necrosis and bile retention were prominent. PCV-2 DNA was identified in ocular, fecal, and nasal secretions. Terminal sera contained antibodies to PPV (4/4) and PCV-2 (3/ 4). Production of PMWS in gnotobiotic swine appears to require PCV-2 and additional infectious agents such as PPV for full disease expression in gnotobiotic piglets.     

Persistence of Escherichia coli O157:H7 in experimentally infected swine

These experiments determined the ability of Escherichia coli O157:H7 to colonize and persist in pigs simultaneously inoculated with other pathogenic E. coli strains. Three-months-old pigs were inoculated with a mixture of five E. coli strains. The mixture included two Shiga toxigenic E. coli (STEC) O157:H7 strains, two enterotoxigenic E. coli (ETEC) strains and one enteropathogenic E. coli (EPEC) strain. A high dose mixture with all five strains at 10(10)CFU/animal (CFU: colony forming units) and a low dose mixture with the STEC strains at 10(7)CFU and the EPEC and ETEC strains remaining at 10(10)CFU were used. The STEC strains persisted in the alimentary tracts of some pigs at 2 months post-inoculation, following inoculation with both the high and low dose mixtures. When all strains were given at 10(10)CFU (high dose) the STEC strains persisted in greater numbers and in more pigs than did the other E. coli strains. The results demonstrated that persistent colonization (> or =2 months) by E. coli O157:H7 can occur in pigs. These findings were similar to those reported from sheep inoculated with the same mixture of E. coli strains. The results are consistent with reports suggesting that pigs have the potential to be reservoir hosts for STEC O157:H7.     

Reproductive failure associated with porcine parvovirus in an enzootically infected pig herd.

Two outbreaks of porcine parvovirus occurred in a unit of 380 sows, with a subsequent decrease in the size of gilts' litters. Of the 203 gilts and 64 primiparous sows which were seronegative at the time of insemination, 134 gilts and 55 primiparous sows seroconverted during pregnancy. Of the second parity sows nine of 271 were still seronegative at the time of insemination but all nine seroconverted during their third gestation. The gilts that seroconverted during their first pregnancy produced on average 0.9 fewer live piglets than the gilts that did not seroconvert. Second litter sows which seroconverted produced 0.3 fewer piglets than those which did not seroconvert. There were no significant differences between sows which seroconverted or did not seroconvert in the numbers of returns to service, or in the percentages of piglets which were born dead or died before weaning.     

Vaccination of swine with an inactivated porcine parvovirus vaccine in the presence of passive immunity

A study was conducted to determine whether low hemagglutination inhibiting (HI) titers (1:5) for porcine parvovirus (PPV) block the development of immune response to a PPV vaccine. Pigs with low (1:5), medium (1:10 or 1:20), or high (1:40 or 1:80) titers were obtained by IV injections with various amounts of PPV immune serum. Pigs were inoculated with 1 or 2 doses of vaccine and were monitored for serum HI antibodies to PPV. Pigs with low titers responded to vaccine just as well as did the seronegative pigs. The HI titers of pigs with medium titers did not increase after first vaccination. After the second vaccination, however, their titers increased and were similar to those of pigs with low titers. High titers blocked the response to vaccination. The pigs that received 2 doses of vaccine had higher titers than did those of pigs that received 1 dose of vaccine. The results indicated that low titers, which would be expected in gilts at the time of vaccination, do not interfere with immunization by the inactivated PPV vaccine, and that 2 doses of vaccine may provide better and longer lasting immune response to inactivated PPV vaccine and probably longer lasting immunity against PPV-induced reproductive failure.     

一例猪细小病毒病的诊断

从取自某猪场表现初产母猪流产胎儿的肾脏、脾脏、肠系膜淋巴结组织,进行研磨后接种猪原代肾细胞,成功分离到一株病毒。该病毒的豚鼠红细胞血凝活性为27,用针对猪细小病毒结构蛋白VP2的特异性引物对分离病毒进行扩增,将扩增结果进行克隆测序,结果经BLAST分析后,证实分离到一株猪细小病毒。     

福建省4个地区规模化猪场细小病毒病血清学调查

应用酶联免疫吸附试验方法对福建省4个地区的33个规模化猪场共计667份血清样品进行了猪细小病毒病抗体检测。检测结果总阳性数为475份,平均阳性率为71.2%,其中总强阳性数308份,平均强阳性率为64.8%。结果表明福建省部分地区可能存在猪细小病毒病流行隐患。     

Efficacy of porcine parvovirus vaccines

Three inactivated porcine parvovirus vaccines were tested for efficacy in 66 susceptible gilts. The gilts were challenged with virulent virus on the 40th day of gestation. All the vaccines provided excellent protection against fetal mortality despite insignificant serological responses to one of them. Good protection was obtained with two of the vaccines even when the dose was substantially reduced. Unvaccinated controls had very few viable fetuses.